Anti-tumor Effects Of Nifuroxazide Combined With IDO2-siRNA Delivered By Attenuated Salmonella On Melanoma Bearing Mice

BackgroundMelanoma is one of the deadliest malignant tumor,so it is still important to find new therapies.For a long time,tumor immunotherapy therapy,especially the immunotherapy combined with other therapies,have attracted more and more attention.Indoleamine2,3-dioxygenase2(IDO2),is an enzyme in the kynurenine pathway that is responsible for the degradation of tryptophan and has been implicated in bolstering immune-inhibitory effect.It is highly expressed in melanoma tissues and significantly inhibits the anti-tumor immunity of the body.It is a new target for the treatment of melanoma now.At the same time,Nifuroxazide,as a kind of intestinal bacteriostatic drug,was found to inhibit the expression of Stat3 in the early stage of our research group,with certain anti-tumor effects.Therefore,we inhibit the growth and proliferation of tumors by blocking the expression of negative immune molecule IDO2 at the gene level with the design of new IDO2-mRNA sequences,meanwhile we combined with Nifuroxazide,and the mechanism of the combination therapy was preliminarily explored.ObjectiveExploring the effect and mechanism of attenuated Salmonella carrying IDO2-siRNA combined with Nifuroxazide against melanomaMethodsExperiment in vitro:The optimal RAW264.7 cells were laid out in a six-well plate at a density of 3×10~5/well.After 16h of culture,IDO2-siRNA of different concentrations was added in the six-well plate,and then extracting the cell proteins for 24h and 48h,and the expression of IDO2 protein was detected by Western blot.Experiment in vivo:1.After digestion and resuspended,the B16 cells which in excellent conditions were digested and counted,and 0.1ml(1×10~6)B16 melanoma cells were injected subcutaneously in the right posterior leg approximately 1cm from the back of the mouse.After 7 days of inoculation,the mice were randomly divided into five groups:PBS group,Scramble group,IDO2-siRNA group,Nifuroxazide group,Nifuroxazide+IDO2-siRNA group,and started the following treatment.A:The first course of treatment(7 days after inoculation):the drug treatment group received injection therapy every day(200?g/mouse),the Scramble group,the IDO2-siRNA group,and the Nifuroxazide+IDO2-siRNA group were treated with liquid bacteria on the seventh day,with a one-time infusion of 4×10~5CFU/mouse.B:The second course of treatment(14 days after inoculation):Scramble group,IDO2-siRNA group,Nifuroxazide+IDO2-siRNA group were treated with the same amount of bacteria again.A total of seven days of drug treatment,two times of bacterial solution treatment.7 days after the end of the second course of bacteria-killing treatment,melanoma tissues of mice were isolated and proteins in tumor tissues were extracted respectively.Expressions of IDO2 and various immune molecules in tumor tissues were detected by Western blot.And we use FACS to detect on a tumor-burdened mice spleen about CD4~+and CD8~+lymphocyte subgroup.2.Survival statistics of mice:the daily survival of mice in each group was recorded.and the effects of different treatment methods,especially attenuated salmonella delivering IDO2-siRNA,on survival of melanoma bearing mice were observed.3.Data processing methods and quality control methodsThe data were expressed as mean±standard deviation,and the statistical software used SPSS19.0 for statistical analysis.The differences between the two groups were analyzed by t test,and P<0.05 was considered statistically significant.Three independent experiments were used for quality control in all experiments.Results1.Cell experiments showed that Nifuroxazide significantly inhibited the proliferation of B16 cells and increased cell apoptosis;2.IDO2-siRNA specifically reduced the expression of IDO2 in RAW cells;3.In the animal model of melanoma xenograft,the combination therapy effectively inhibited the growth of tumor in mice and increased the survival rate of tumor bearing mice;4.Combination therapy can inhibit the phosphorylation of Stat3 in B16 cells and inhibit the expression of IDO proteins;5.Compared with other groups,the combined treatment group,HE staining showed a decrease in tumor cell atypia and an increase in apoptotic cells;6.The combination therapy increased the infiltration of T lymphocytes in the tumor tissue;7.The combination treatment effectively increased the ratio of CD4~+T and CD8~+T lymphocytes in spleen of tumor-bearing mice.Conclusion1.The IDO2-siRNA which was independently designed can effectively inhibit the expression of IDO2 in cells.2.The vivo animal experiments showed that IDO2-siRNA combined with Nifuroxazide could play a significant therapeutic effect on tumor-bearing mice,and The antitumor immune response of tumor-bearing mice was enhanced.